Bioluminescence Imaging And Human Neutrophil Kinetics Of Cellular Signal Transduction

Dissipative structure theory has extensively been applied in the research of physics, chemistry, biology and society. Especially, it is being studied in the field of life science including network regulatory of signal transduction, control of immune defence, control of embryonic development, and so on. In this paper, we studied the dynamics of hydrogenated nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and calcium (Ca²⁺) signal transductions in neutrophils with dissipative structure theory.Neutrophils are one of the body's main components of immune system. Dynamics of NAD(P)H and Ca²⁺ signal transductions has been found to exhibit characteristic spatiotemporal patterns in such immunity processes of human neutrophils. Nowadays, the acquisition of spatiotemporal information depends mainly on the technique of living fluorescence imaging. Therefore, we constructed weak light micro-imaging system with high spatiotemporal resolution to study the autofluorescence NAD(P)H in single human neutrophils. The micro-imaging system can investigate fluorescence signals at about the milliseconds range with high spatial resolution. As a result, we achieved real-time, fast and visual imaging of autofluorescence NAD(P)H in single human neutrophils without running into the quenching or the phototoxicity problem due to the weak light micro-imaging system So that it is able to provide potent experimental support for studying spatiotemporal information in the dynamic process of intracellular NAD(P)H signal transduction.On this basis, we detailedly studied the role and signaling pathway of exogenous Nitric oxide (NO) in modulating Cytosolic calcium concentration ([Ca²⁺]_c) of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils. NO, as an immunoregulatory messenger, has been reported that it can execute modulating in a wide variety of physiological functions and signal transductions in neutrophil chemotaxis, adhesion, phagocytosis, apoptosis, and oxidative burst. Meanwhile, PMA, a potent protein kinase C (PKC) activator, can induce respiratory burst of neutrophils by activating PKC, which subsequently activates NADPH Oxidase. To the best of our knowledge it is the first demonstration that exogenous NO induced calcium release from intracellular IP₃ receptor-sensitive stores and extracellular calcium influx via Snitrosylation reaction, but not via the activation of the NO-sGC-cGMP signaling pathway. More importantly, the effect of SNP on neutrophil [Ca²⁺]_c was dependent on the PMA-induced respiratory burst. Therefore, this study on modulation of neutrophils [Ca²⁺]_c by exogenous NO might be much more useful in the research of immunological disease. Meanwhile, it provides an ideal model to the research of dissipative patterns of [Ca²⁺]_c in neutrophils.In addition, this dissertation studied cytoskeleton change in rheumatoid arthritis (RA) rat Synoviocytes induced by PMA via the imaging of atomic force microscopy. The mechanism of RA correlates closely to the abnormal proliferation of neutrophils. PMA is usually used to simulate process of inflammation. The results showed that synoviocyte cytoskeleton changed significantly after PMA activation. The results also indicated that the activation of PKC might play crucial role in cytoskeleton changing of synoviocytes, which could help us to understand the mechanism of RA.In conclusion, we have made in-depth study of dynamic process of NAD(P)H and Ca²⁺ signal transductions in human neutrophils with our fluorescence micro-imaging system. The results are able to provide a new perspective for understanding the mechanism of immune response and dissipative patterns of body's immune defense system.